Porous Polymeric Articles

ABSTRACT

Porous polymeric articles, and more specifically, porous polymeric articles for tissue engineering and organ replacement, are described. In some embodiments, methods described herein include use of a polymer-solvent system (e.g., phase inversion) to generate porosity in a structure. The process may include formation of a structure precursor material including a first crosslinkable component and a second component that can be precipitated in a precipitation medium. The structure precursor material may be shaped into a three-dimensional shape by a suitable technique such as three-dimensional printing. Upon shaping of the structure precursor material, at least a portion of the first component may be crosslinked. The structure may then be contacted with a precipitation medium to remove the precursor solvent from the structure, which can cause the second polymer component to precipitate and form a porous structure containing a network of uniform pores. In some embodiments, the porous structure is constructed and arranged for use as a template for ultrafiltration, cell growth, and/or for forming complex, biomimetic, porous biohybrid organs, where living cells can be immobilized and perform their normal physiological functions.

FIELD OF INVENTION

The present invention relates generally to porous polymeric articles,and more specifically, to porous polymeric articles for tissueengineering and organ replacement.

BACKGROUND

Tissue engineering and organ transplantation are principally concernedwith the replacement of tissue and organs that have lost function due toinjury or disease. In one approach toward this goal, organs aretransplanted into a patient. However, the side effects oftransplantation can be unpleasant, and can compromise the health of theorgan recipient. In another approach, cells are cultured in vitro onbiodegradable polymeric scaffolds to form tissues or neo organs that arethen implanted into the body at the necessary anatomical site.

Several techniques have been proposed for forming scaffolds for tissuegrowth. For instance, U.S. Patent Publication No. 2002/0182241, entitled“Tissue Engineering of Three-Dimensional Vascularized UsingMicrofabricated Polymer Assembly Technology,” by Borenstein et al.,describes two-dimensional templates that are fabricated usinghigh-resolution molding processes. These templates are then bonded toform three-dimensional scaffold structures with closed lumens. U.S. Pat.No. 6,176,874, entitled “Vascularized Tissue Regeneration MatricesFormed by Solid Free Form Fabrication Techniques,” by Vacanti et al.,describes solid free-form fabrication methods used to manufacturedevices for allowing tissue regeneration and for seeding and implantingcells to form organ and structural components. U.S. Patent PublicationNo. 2003/0069718, entitled “Design Methodology for Tissue EngineeringScaffolds and Biomaterial Implants,” by Hollister et al., describesanatomically shaped scaffold architectures with heterogeneous materialproperties, including interconnecting pores.

Despite the above efforts, significant developments in connection withmany internal, physical structures, especially those of hollow andepithelial organs, has been limited, and improvements are needed.Particularly, new methods for fabricating articles having small anduniform pore sizes for tissue engineering and organ replacement would bebeneficial.

SUMMARY OF THE INVENTION

Porous polymeric articles, and more specifically, porous polymericarticles for tissue engineering and organ replacement are provided. Inone aspect, a series of methods of fabricating a structure for use as atemplate for cell growth are provided. In one embodiment, the methodcomprises dissolving at least first and second polymer components in aprecursor solvent to form a structure precursor material, shaping thestructure precursor material into a structure suitable for use as atemplate for cell growth, crosslinking the first polymer component, andremoving at least a portion of the precursor solvent from the structure,thereby forming a plurality of pores in the structure.

In another embodiment, a method of fabricating a structure for use as atemplate for cell growth is provided. The method comprises providing astructure precursor material comprising at least first, second, andthird components, shaping the structure precursor material into astructure suitable for use as a template for cell growth, crosslinkingthe first component, precipitating the second component in aprecipitation medium, and removing the third component from thestructure in the precipitation medium, thereby forming a plurality ofpores in the structure.

In another embodiment, a method of fabricating a structure for use as atemplate for cell growth is provided. The method comprises mixing atleast first and second polymer components in a precursor solvent to forma homogeneous structure precursor material, wherein the first and secondpolymer components and the precursor solvent are miscible at 25 degreesCelsius and 1 atm, printing the structure precursor material to form athree-dimensional structure suitable for use as a template for cellgrowth, and removing the precursor solvent from the structure, therebyforming a plurality of pores in the structure.

In another embodiment, a method of fabricating a structure for use as atemplate for cell growth is provided. The method comprises forming acell growth template precursor structure comprising at least first andsecond polymer components and a fluid carrier, crosslinking the firstpolymer component thereby forming a self-supporting structure, andremoving at least a portion of the fluid carrier from theself-supporting structure, thereby forming a plurality of pores in thestructure suitable for templated cell growth, wherein the porousstructure is formed in a shape suitable for templated cell growth.

In another embodiment, a method of fabricating a structure for use as atemplate for cell growth is provided. The method comprises dissolving atleast first and second polymer components in a precursor solvent to forma structure precursor material, shaping the structure precursor materialinto a structure suitable for use as a template for cell growth,exposing the structure precursor material to UV radiation, and removingat least a portion of the precursor solvent from the structure, therebyforming a plurality of pores in the structure.

In another aspect, an article for use as a template for cell growth isprovided. The article comprises a structure comprising at least one walldefining a cavity, and a plurality of pores having an average pore sizeof less than or equal to 20 microns formed in at least a portion of thewall, wherein no more than about 5% of all pores deviate in size fromthe average pore size of the plurality of pores by more than about 20%,wherein the structure is constructed and arranged for use as a templatefor cell growth.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 shows a process for forming a three-dimensional structureaccording to one embodiment of the invention;

FIGS. 2A and 2B show schematic diagrams of a three-dimensional printingprocess for forming three-dimensional structures, according to oneembodiment of the invention;

FIG. 3 shows a schematic diagram of a phase inversion process forforming a porous structure, according to one embodiment of theinvention;

FIG. 4 shows a schematic diagram of a filtration process forcharacterizing pore size and molecular weight cutoff of a porousstructure, according to one embodiment of the invention;

FIG. 5 shows a plot of water flux as a function of pressure for variousporous membranes, according to one embodiment of the invention;

FIG. 6A-6F shows SEM micrographs of porous PS-FC membranes, according toone embodiment of the invention;

FIG. 7 shows PEG rejection curves for the porous membranes of FIGS.6A-6F as a function of PEG molecular weight, according to one embodimentof the invention;

FIG. 8A shows MDCK cells adhered to a commercial membrane without theuse of adhesion proteins, according to one embodiment of the invention;and

FIGS. 8B-8C show MDCK cells adhered to a PS-FC membrane without the useof adhesion proteins, according to one embodiment of the invention.

DETAILED DESCRIPTION

Porous polymeric articles, and more specifically, porous polymericarticles for tissue engineering and organ replacement, are described. Insome embodiments, methods described herein include use of apolymer-solvent system (e.g., phase inversion) to generate porosity in astructure. The process may include formation of a structure precursormaterial including a first crosslinkable component and a secondcomponent that can be precipitated in a precipitation medium. Thestructure precursor material may be shaped into a three-dimensionalshape by a suitable technique such as three-dimensional printing. Uponshaping of the structure precursor material, at least a portion of thefirst component may be crosslinked. The structure may then be contactedwith a precipitation medium to remove the precursor solvent from thestructure, which can cause the second polymer component to precipitateand form a porous structure containing a network of uniform pores. Insome embodiments, the porous structure is constructed and arranged foruse as a template for ultrafiltration, cell growth, and/or for formingcomplex, biomimetic, porous biohybrid organs, where living cells can beimmobilized and perform their normal physiological functions.

Advantageously, structures described herein may have attractivebiofunctional characteristics such as uniform pores having a sharpmolecular weight cut-off (MWCO), high filtration and diffusion fluxes,and good mechanical strength, biocompatibility and formability.

Although much of the description herein involves an exemplaryapplication of the present invention related to using porous polymericarticles as scaffolds for tissue engineering and/or organ replacement,the invention and its uses are not so limited, and it should beunderstood that the invention can also be used in other settings such asfor filtration, purification, and separation processes.

In some embodiments, structures described herein can be drawn, imaged,and/or scanned using a variety of tools, including computer-aided design(CAD) tools, high-resolution multi-section computed tomography (CT)scans, and/or three-dimensional scanners. For instance, for structuresto be used for tissue engineering and/or organ replacement, a CT scan ofa tissue and/or organ of a patient can be converted into a proper fileformat, and fed into a system that can produce the structures. A varietyof techniques can be used to form the structures, as described in moredetail below. These methods can, in some cases, control compositions andmicro-architectures of the structures. Appropriate systems andtechniques for fabricating structures for tissue engineering and/ororgan replacement include, but are not limited to, three-dimensionalprinting (e.g., three-dimensional layering), multi-photon lithography,stereolithography (SLA), selective laser sintering (SLS) or laserablation, ballistic particle manufacturing (BPM), laminated objectmanufacturing, and fusion deposition modeling (FDM). In certainpreferred embodiments, structures are formed by three-dimensionalprinting. Other techniques for fabricating structures for tissueengineering and/or organ replacement can also be used. Such techniquescan be combined with appropriate materials and/or steps to fabricateporous articles described herein.

In one embodiment, a three-dimensional printing technique is used tofabricate a porous article. The three-dimensional printing technique mayinclude the use of a tool such as the Eden 260 Rapid Prototyping Tool(RPT). The Eden 260 RPT is a polymer dispensing system that can printdroplets of a polymer precursor material and, if desired, a sacrificialmaterial, using a piezoelectric-actuated nozzle. Using such tools, athree-dimensional image file can be processed and the image may besliced into many layers. Each layer can then printed on top of eachother, and at least a portion of the polymer precursor material can bepolymerized and/or crosslinked.

An example of a process for forming a three-dimensional structure isshown in FIG. 1. As shown in process 6, structure precursor material 16to be shaped (e.g., into a template for cell growth) may include firstcomponent 8 comprising a monomer (e.g., a UV crosslinkable monomer) andsecond component 10 comprising a monomer/polymer that can beprecipitated. The first and/or second components may be dissolved insolvent 12 (e.g., a third component or a fluid carrier). In someembodiments, step 14 of combining (e.g., mixing) the first and secondcomponents with the solvent may form a homogeneous solution. Thestructure precursor material can then be shaped in step 18 into a firstprecursor structure 20, which may be a cell growth template precursorstructure. The shaping of the precursor structure material may takeplace by three-dimensional printing, as illustrated in FIGS. 2A and 2B,or by another suitable technique. For example, in the embodimentillustrated in FIGS. 2A and 2B, the structure precursor material (and asacrificial material, if desired) can be dispensed droplet by dropletand layer by layer. Tools 40 and 42 can dispense droplets 44 of the sameor different materials using one or more nozzles 46. Droplets 44 may beprinted to form first precursor structure 20, supported by substrateholder 48. As shown in FIG. 2A, first precursor structure 20 can beformed by a vertical printing process; FIG. 2B shows horizontal printingof first precursor structure 20. Optionally, after each layer ofmaterial has been dispensed, a roller may be used to smooth out thesurface. The first component of the structure precursor material (e.g.,the crosslinkable monomer) may be polymerized (and/or crosslinked) instep 22 of FIG. 1, which may include, for example, exposure of thestructure precursor material to UV radiation or any suitable source thatcan cause polymerization and/or crosslinking of at least a portion ofthe material. This process may be repeated until the formation of secondprecursor structure 24, which may be in the form of a solid orsemi-solid structure (e.g., a self-supporting structure). In otherembodiments, several or all layers of the structure precursor materialmay be dispensed before polymerization and/or crosslinking of at leastone component of the structure precursor material. In some cases, thesecond component does not substantially polymerize and/or crosslink uponexposure to UV radiation. The structure can then be contacted with aprecipitation medium in step 26 and at least a portion of the precursorsolvent may be removed in the medium. This process can cause the secondcomponent to precipitate and form porous structure 28 containing anetwork of uniform pores. Alternatively, in some embodiments, firstprecursor structure 20 may be contacted with a precipitation medium instep 26, followed by polymerization and/or crosslinking of a componentof the precursor structure to form porous structure 28. The porousstructure may be formed in a shape suitable for templated cell growth.

In some cases, porous structure 28 may be designed to include open areas(e.g., pores and/or cavities). During fabrication, the open areas may befilled with a sacrificial material. The sacrificial material and theprecursor material may be dispensed by separate nozzles of athree-dimensional printer. After printing, the sacrificial material maybe removed, for example, by dissolving the material in a solvent.Typically, a suitable sacrificial material includes one that is solublein a solution that does not dissolve the structure precursor material.In some cases, the sacrificial material is not polymerizable and/orcrosslinkable; however, in other cases, the sacrificial material ispolymerizable and/or crosslinkable.

A further description of the phase inversion process is now described.As described herein, in some embodiments, fabrication of a structure caninvolve dissolving one or more components (e.g., a first and/or a secondpolymer component, which may include monomers and/or polymers) in anappropriate precursor solvent to form a structure precursor solution. Inone particular embodiment, the first and second components are miscibleat 25 degrees Celsius and 1 atm. The structure precursor solution can beshaped into the desired structure. For example, in one embodiment, thestructure precursor solution is cast as a membrane or hollow fiber. Inanother embodiment, the structure precursor solution is shaped into athree-dimensional structure by a suitable technique, such asthree-dimensional printing. Optionally, at least one component (e.g., afirst polymer component) of the structure can be polymerized and/orcrosslinked, e.g., by exposing the component to ultraviolet (UV)radiation. Polymerization and/or crosslinking may take place after all,or portions, of the structure has been shaped. In some cases,polymerization and/or crosslinking can cause solidification of portionsof the structure. The structure can then be immersed in a precipitationmedium (e.g., a solvent, also called a “non-solvent”, in the form of aliquid or a gas) that can precipitate at least one component (e.g., asecond polymer component) of the structure. This process can causeseparation of the precursor structure into a solid polymer and a liquidsolvent phase including the precursor solvent. At least a portion of theprecursor solvent may be removed in the precipitation medium, which cancause the second polymer component to precipitate and form a porousstructure containing a network of uniform pores.

Parameters that affect the structure and properties of the desiredstructure may include the composition of the precipitation media, thecomponent concentrations, the viscosity of the precursor material(which, in turn, may depend on the method used to shape/form thestructure), the relative glass transition temperature and the viscosityratio (or molecular weight ratio) of the components (e.g., if theprecursor material includes more than one components), the temperatureof the structure precursor material and/or precipitation medium,component molecular weight and solubility parameters (of thecomponent(s), solvent, and precipitation medium), and the amount andtype of precursor solvent. These factors can be varied to producestructures with, for example, a large range of pore sizes (e.g., from0.01 to 20 microns), and, in some embodiments, with no more than about5% of all pores deviating in size from the average pore size by morethan about 20%, as described in more detail below.

Different methods of precipitation can be used to induce precipitationof a component of a structure precursor material. In some instances,precipitation of a component from a precursor structure can be caused bychanging the concentration of the component in the precursor structure.For instance, in one embodiment, a precursor structure or precursorsolution comprising a polymer component and a precursor solvent can bebrought into contact with a precipitation medium. At least a portion ofthe precursor solvent can diffuse outwards into the precipitation mediumand at least a portion of the precipitation medium can diffuse into thestructure or precursor solution. After a given period of time, theexchange of the precursor solvent and precipitation medium can cause theprecursor structure/solution to become thermodynamically unstable. As aresult, demixing can occur and the polymer component can precipitate toform a solid network. Alternatively, in some cases the precipitationmedium may be a gas (e.g., air, nitrogen, oxygen, and carbon dioxide)and evaporation of the precursor solvent can cause precipitation of apolymer component. In another embodiment, a polymer component dissolvedin a solvent can solidify by a temperature change (e.g., upon cooling).This may be performed, for example, by reducing the temperature of theprecursor structure/solution to below the glass transition temperatureor the melting point of the polymer component to be precipitated.

The rate of precipitation of one component of the structure precursormaterial may be controlled by choosing appropriate compositions and/orconditions of the precipitation medium. For instance, it is known thatthe quicker a component is caused to precipitate, the finer is thedispersion of the precipitating phase. High rates of precipitation mayoccur by exposing the precipitating component to a precipitation mediumhaving a very different solubility parameter than that of the component.The length of exposure of the component to the precipitation medium andthe temperature difference between the two may also change the rate ofprecipitation. Accordingly, structural integrity and morphologicalproperties of the final porous structure can be varied by controllingsuch parameters.

Materials suitable for use as a precipitation medium include, forexample, liquids or gases that can cause at least one component of thestructure precursor material to precipitate upon exposure to the medium.For example, a structure precursor material comprising polysulfone canbe precipitated by exposure of the material to water, which acts as asuitable precipitation medium. A suitable precipitation medium for astructure precursor material may be chosen based on the solubility ofthe component to be precipitated in the precipitation medium, e.g.,using known solubility properties of the materials or by simpleexperimentation. For instance, solubility parameters (e.g., Hildebrandparameters), as described in Barton, Handbook of Solubility Parameters,CRC Press, 1983, may be used to determine the likelihood of solubilityof one component in another. Typically, chemical components havingdifferent values of solubility parameter are not soluble in one another.In certain embodiments, a structure precursor material component that isnon reactive with, and precipitates upon exposure to, a precipitationmedium is preferred. Accordingly, a structure precursor materialcomponent and a precipitation medium having different values ofsolubility parameter may be chosen. Those of ordinary skill in the artcan also choose an appropriate structure precursor material componentand/or precipitation medium by a simple screening test. One simplescreening test may include mixing the structure precursor materialcomponent with the precipitation medium and determining whether thecomponents react with and/or causes the precursor material component toprecipitate. Varying conditions such as temperatures and concentrationsof materials may be used in such an experimentation. Other simple testscan be conducted by those of ordinary skill in the art.

A variety of materials can be used to fabricate structures of thepresent invention. Materials used to form structures for tissueengineering and/or organ replacement may be biocompatible, and caninclude, for example, synthetic or natural polymers, inorganicmaterials, or composites of inorganic materials with polymers.

As described above, in some embodiments, structures described herein areformed of a structure precursor material that includes at least firstand second polymer components. The first and second polymer componentsmay be, for example, monomers that can be or a polymerized and/orcrosslinked, or polymers that can be further polymerized and/orcrosslinked by any suitable means. Sometimes, the first polymercomponent can be dissolved in the second polymer component (or in asolvent compatible with both polymer components) such that the componentmolecules interpenetrate one another. The structure precursor materialmay not stable thermodynamically, meaning that a demixing process mayoccur in the material. To increase the stability, it is often necessaryto polymerize, crosslink or precipitate one or both polymer components.Accordingly, in one embodiment, a polymer component of a structureprecursor material is substantially soluble in a precursor solvent butsubstantially insoluble in a precipitation medium, such that at least aportion of the component precipitates upon contact with theprecipitation medium. In another embodiment, a polymer component of astructure precursor can be polymerized and/or crosslinked by a suitabletechnique, such as exposure to UV radiation, heat, and or a crosslinkingagent. In yet another embodiment, polymer components (and/or a precursorsolvent) are chosen at least partially based on their solubility in oneanother, e.g., using known solubility properties of the components or bysimple experimentation. For instance, solubility parameters (e.g.,Hildebrand parameters), as described in Barton, Handbook of SolubilityParameters, CRC Press, 1983, may be used to determine the likelihood ofsolubility of one component in another. Typically, chemical componentshaving similar values of solubility parameter are soluble in oneanother. Those of ordinary skill in the art can also choose anappropriate polymer and/or solvent by, e.g., the likelihood ofreactivity between the components and the solvent, and/or by a simplescreening test. One simple screening test may include mixing the polymercomponents together, optionally with a precursor solvent, anddetermining whether the components react with one another and/or form ahomogeneous solution. In certain embodiments, non-reactive componentsthat form a miscible (e.g., homogeneous) solution at 25 degrees Celsiusand 1 atm are preferred. Other simple tests can be conducted by those ofordinary skill in the art.

In some embodiments, a polymer component includes one or morephotocurable (e.g., crosslinkable) polymers (or monomers). For instance,photocurable polymers may include ultra-violet or visible-light curablepolymers. Particular materials include photocurable acrylic monomers,acrylic polymers, UV curable monomers, thermal curable monomers, polymersolutions such as melted polymers and/or oligomer solutions, poly methylmethacrylate, poly vinylphenol, benzocyclobutene, polyethylene oxideprecursors terminated with photo-crosslinking end groups, one or morepolyimides, and monomers of such polymers. In some cases, acrylate-basedphoto-polymers can include one or more components such as a sensitizerdye, an amine photo-initiator, and a multifunctional acrylate monomer.For example, pentaerythritol triacrylate (PETIA,) can form the backboneof the polymer network, N-methyldiethanolamine (MDEA) can be used as aphoto-initiator, and Eosin Y (2-, 4-, 5-, 7-tetrabromofluoresceindisodium salt) can be used as a sensitizer dye. This system isparticularly sensitive in the spectral region from 450 to 550 nm, andcan be used, for instance, in two-photon lithography involving a 1028 nmlaser. In another example, an organic-inorganic hybrid such as ORMOCER®(Micro Resist Technology) can be used to fabricate structures describedherein. This material can show high transparency in the visible and nearinfrared ranges, can contain a highly crosslinkable organic network, canincorporate inorganic components that may lead to high optical qualityand high mechanical and thermal stability, and can be biocompatible forcertain types of cells and/or cellular components. In yet anotherexample, acrylate and epoxy polymers such as ethoxylatedtrimethylolpropane triacrylate ester and alkoxylated trifunctionalacrylate ester can be used to form structures.

Structure precursor materials may additionally include one or morephotoinitiators and/or crosslinkers for polymerization and/orcrosslinking. Additionally, the structure precursor material mayoptionally be diluted in one or more solvents in order to decrease theviscosity of the material and to make it suitable for application, forexample, in an ejection mechanism such as a three-dimensional printer.

In certain embodiments, photopolymerizable materials that are alsobiocompatible and water-soluble can be used to form structures fortissue engineering and/or organ replacement. A non-limiting exampleincludes polyethylene glycol tetraacrylate, which can bephotopolymerized with an argon laser under biologically compatibleconditions, i.e., using an initiator such as triethanolamine,N-vinylpyrrolidone, and eosin Y. Similar photopolymerizable units havinga poly(ethylene glycol) central block, extended with hydrolyzableoligomers such as oligo(d,l-lactic acid) or oligo(glycolic acid), andterminated with acrylate groups, may be used. Other polymerizable and/orcrosslinkable polymers that polymerize or crosslink, for example, uponexposure to heat and/or chemical crosslinking agents may also be used.

Additional examples of polymer components that can be used to formstructures described herein include but are not limited to: polyvinylalcohol, polyvinylbutryl, polyvinylpyridyl, polyvinyl pyrrolidone,polyvinyl acetate, acrylonitrile butadiene styrene (ABS),ethylene-propylene rubbers (EPDM), EPR, chlorinated polyethylene (CPE),ethelynebisacrylamide (EBA), acrylates (e.g., alkyl acrylates, glycolacrylates, polyglycol acrylates, ethylene ethyl acrylate (EEA)),hydrogenated nitrile butadiene rubber (HNBR), natural rubber, nitrilebutadiene rubber (NBR), certain fluoropolymers, silicone rubber,polyisoprene, ethylene vinyl acetate (EVA), chlorosulfonyl rubber,flourinated poly(arylene ether) (FPAE), polyether ketones, polysulfones,polyether imides, diepoxides, diisocyanates, diisothiocyanates,formaldehyde resins, amino resins, plyurethanes, unsaturated polyethers,polyglycol vinyl ethers, polyglycol divinyl ethers, poly(anhydrides),polyorthoesters, polyphosphazenes, polybutylenes, polycapralactones,polycarbonates, and protein polymers such as albumin, collagen, andpolysaccharides, copolymers thereof, and monomers of such polymers. Incertain embodiments, a polymer component is chosen based on itscompatibility with a three-dimensional printing technique.

In one particular embodiment, a structure precursor material comprisesthe UV curable acrylic monomer comprising Objet FullCure™ 3D printingbuild material, which is available from Objet Geometries Inc. Uponexposure of a structure precursor material comprising the build materialto UV radiation, at least a portion of the acrylic monomers maypolymerize and/or crosslink to form a solid or semi-solid precursorstructure. In this embodiment, the structure precursor material mayadditionally comprise a photoinitiator for polymerization, and may bediluted in one or more solvents, such as an alcohol, e.g., isopropropylalcohol, ethanol, and/or methanol, or any other suitable solvent, inorder to decrease the UV curable monomer viscosity and to make itsuitable for application, for example, in an ejection mechanism such asa three-dimensional printer.

In some cases, a polymer precursor material includes a polymer that canprecipitate upon exposure to a precipitation medium. In one particularembodiment, the polymer component is a polysulfone. Polysulfonesinclude, for example, polyether sulfones, polyaryl sulfones (e.g.,polyphenyl sulfone), polyalkyl sulfones, polyaralkyl sulfones, and thelike.

Other polymers that may precipitate upon exposure to a precipitationmedium that may be used as a structure precursor component include, butare not limited to, polyamines (e.g., poly(ethylene imine) andpolypropylene imine (PPI)); polyamides (e.g., polyamide (Nylon),poly(ε-caprolactam) (Nylon 6), poly(hexamethylene adipamide) (Nylon66)), polyimides (e.g., polyimide, polynitrile, andpoly(pyromellitimide-1,4-diphenyl ether) (Kapton)); vinyl polymers(e.g., polyacrylamide, poly(2-vinyl pyridine), poly(N-vinylpyrrolidone),poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(vinylacetate), poly (vinyl alcohol), poly(vinyl chloride), poly(vinylfluoride), poly(2-vinyl pyridine), vinyl polymer, polychlorotrifluoroethylene, and poly(isohexylcynaoacrylate)); polyacetals; polyolefins(e.g., poly(butene-1), poly(n-pentene-2), polypropylene,polytetrafluoroethylene); polyesters (e.g., polycarbonate, polybutyleneterephthalate, polyhydroxybutyrate); polyethers (poly(ethylene oxide)(PEO), poly(propylene oxide) (PPO), poly(tetramethylene oxide) (PTMO));vinylidene polymers (e.g., polyisobutylene, poly(methyl styrene),poly(methylmethacrylate) (PMMA), poly(vinylidene chloride), andpoly(vinylidene fluoride)); polyaramides (e.g., poly(imino-1,3-phenyleneiminoisophthaloyl) and poly(imino-1,4-phenylene iminoterephthaloyl));polyheteroaromatic compounds (e.g., polybenzimidazole (PBI),polybenzobisoxazole (PBO) and polybenzobisthiazole (PBT));polyheterocyclic compounds (e.g., polypyrrole); polyurethanes; phenolicpolymers (e.g., phenol-formaldehyde); polyalkynes (e.g., polyacetylene);polydienes (e.g., 1,2-polybutadiene, cis or trans-1,4-polybutadiene);polysiloxanes (e.g., poly(dimethylsiloxane) (PDMS),poly(diethylsiloxane) (PDES), polydiphenylsiloxane (PDPS), andpolymethylphenylsiloxane (PMPS)); and inorganic polymers (e.g.,polyphosphazene, polyphosphonate, polysilanes, polysilazanes); monomersof such polymers, as well as other polymers and monomers describedherein.

Structures described herein may be hydrophobic or hydrophilic.Hydrophobic structures can be formed of hydrophobic polymers including,but not limited to, polypropylene, polyvinylidene fluoride,polyethylene, polyvinylidene fluoride, poly(tetrafluoroethylene). Insome cases, at least a portion of a hydrophobic can be made hydrophilic,e.g., by surface modification. Hydrophilic polymers may also be used, asdescribed herein.

A polymer component may be non-biodegradable or biodegradable (e.g., viahydrolysis or enzymatic cleavage). In some embodiments, biodegradablepolyesters such as polylactide, polyglycolide, and other alpha-hydroxyacids can be used to form structures. By varying the monomer ratios, forexample, in lactide/glycolide copolymers, physical properties anddegradation times of the polymer can be varied. For instance,poly-L-lactic acid (PLLA) and poly-glycolic acid (PGA) exhibit a highdegree of crystallinity and degrade relatively slowly, while copolymersof PLLA and PGA, PLGAs, are amorphous and rapidly degraded.

In some cases, biocompatible polymers having low melting temperaturesare desired. Non-limiting examples include polyethylene glycol (PEG) 400(melting temperature=4-80° C.), PEG 600 (melting temperature=20-25° C.),PEG 1500 (melting temperature=44-480° C.), and stearic acid (meltingtemperature=70° C.).

In some embodiments, a polymer precursor material can include anon-polymeric material. Non-limiting examples of such materials includeorganic and inorganic materials such as ceramics, glass, hydroxyapatite,calcium carbonate, buffering agents, as well as drug delivery carriers(e.g., gels), which can be solidified by application of an adhesive orbinder.

In certain embodiments, additives can be added to a structure precursormaterial. Additives may, for instance, increase a physical (e.g.,strength) and/or chemical property (e.g., hydrophilicity/hydrophobicity)of the material in which the structure is formed. Additives can bedispersed throughout the structure precursor material and/or can beincorporated within certain region(s) of a structure. In some cases,additives can be incorporated during formation of the structure by athree-dimensional fabrication process; in other cases, additives can beincorporated into the structure after the overall shape of the structurehas been formed. Additives can also be incorporated into and/or onto astructure by adsorption or by chemically reacting the additive onto thesurface of the polymer, i.e., by coating or printing the additive ontothe structure. Non-limiting examples of additives include bioactiveagents (e.g., therapeutic agents, proteins and peptides, nucleic acids,polysaccharides, nucleic acids, and lipids, including anti-inflammatorycompounds, antimicrobial compounds, anti-cancer compounds, antivirals,hormones, antioxidants, channel blockers, and vaccines), surfactants,imaging agents, and particles. If desired, additives may be processedinto particles using spray drying, atomization, grinding, or otherstandard techniques. In some cases, additives can be formed intoemulsifications, micro- or nano-particles, liposomes, or other particlesthat can be incorporated into the material of a structure. In someembodiments, composite structures for tissue engineering and/or organreplacement can be formed by combining inorganic and organic components.Particles incorporating an additive can have various sizes; for example,particles may have a cross-sectional dimension of less than 1 mm, lessthan 100 microns, less than 50 microns, less than 30 microns, less than10 microns, less than 5 microns, less than 1 micron, less than 100nanometers, or less than 10 nanometers.

In some cases, it is desirable to release an additive from portions of astructure when the structure is in its environment of use (e.g.,implanted in a mammalian body). Release of an additive may includehydrolysis and/or degradation of the polymer forming the structure. Therelease rate of the additive can be determined, in some instances, bythe degradation rate of the polymer. The release rate of the additivecan be controlled by the distribution of the additive throughout thepolymer and/or by variation of the polymer microstructure (e.g., densityof the polymer) such that the degradation rate varies with certainportions of the structure.

A structure precursor material described herein may have any suitableviscosity to make it compatible with a three-dimensional fabricationtechnique. In some embodiments, the viscosity of the precursor structurematerial is between 1-1,000, centipoise (cps), between 1,000-2,000 cps,between 2,000-5,000 cps, between 5,000-10,000 cps, between 10,000-15,000cps, between 15,000-20,000 cps, between 20,000-25,000 cps, between25,000-30,000 cps, between 30,000-35,000 cps, between 35,000-40,000 cps,between 40,000-45,000 cps, or between 45,000-50,000 cps. In certainembodiments, the viscosity of the precursor structure material isgreater than 10,000 cps, greater than 20,000 cps, greater than 30,000cps, greater than 40,000 cps, greater than 50,000 cps, or greater than60,000 cps. Viscosity of the structure precursor material may bedecreased by various means such as my adding a diluent to the materialand/or increasing the temperature of the material. The viscosity of thematerial may be increased by various means such as by adding a filler(e.g., particles) or a viscous fluid to the material and/or decreasingthe temperature of the material.

A component of a structure precursor material described herein may haveany suitable molecular weight. In some cases, a component has amolecular weight between 10-100 g/mol, between 100-1,000 g/mol, between1,000-5,000 g/mol, between 5,000-10,000 g/mol, between 10,000-15,000g/mol, between 15,000-20,000 g/mol, between 20,000-25,000 g/mol, between25,000-30,000 g/mol, between 30,000-35,000 g/mol, between 35,000-40,000g/mol, between 40,000-45,000 g/mol, or between 45,000-50,000 g/mol.Components having a molecular greater than 50,000 g/mol can also beused.

Any suitable molecular weight ratio of polymerizable/crosslinkable andprecipitating components can be used in structure precursor materialsdescribed herein. For example, the molecular weight ratio of a first,polymerizable and/or crosslinkable component to a second, precipitatingcomponent greater than or equal to 0.01:1, greater than or equal to0.05:1, greater than or equal to 0.1:1, greater than or equal to 0.2:1,greater than or equal to 0.4:1, greater than or equal to 0.6:1, greaterthan or equal to 0.8:1, greater than or equal to 1:1, greater than orequal to 1.2:1, greater than or equal to 1.5:1, greater than or equal to2:1, greater than or equal to 3:1, greater than or equal to 5:1, greaterthan or equal to 10:1, or greater than or equal to 20:1.

As described herein, in some embodiments, structure precursor materialsare designed to have certain weight ratios of a first component and asecond component. The first component may be a material that can bepolymerized and/or crosslinked and the second component may be amaterial that can precipitate upon exposure to a precipitation medium.The ratio of the two components can change the physical properties(e.g., hardness) of the final porous structure. Additionally, in somecases the pore size of the final porous structure can be varied bychanging the ratio of the components. For instance, in some embodiments,structure precursor materials including an increasing concentration of apolysulfone precipitating component relative to a FullCure™polymerizable/crosslinkable component can result in smaller pore sizes.Accordingly, the weight ratio of a precipitating component to apolymerizable/crosslinkable component in a structure precursor materialmay vary depending on the desired pore size in the final porousstructure, and may be, for example, greater than or equal to 0.2:1,greater than or equal to 0.4:1, greater than or equal to 0.6:1, greaterthan or equal to 0.8:1, greater than or equal to 1:1, greater than orequal to 1.2:1, greater than or equal to 1.5:1, greater than or equal to2:1, greater than or equal to 3:1, greater than or equal to 5:1, greaterthan or equal to 8:1, greater than or equal to 10:1, greater than orequal to 15:1, or greater than or equal to 20:1.

The “pore size” of a structure refers to the length of the shortest line(e.g., cross-sectional dimension) parallel to a surface of the structureconnecting two points around the circumference of a pore and passingthrough the geometric center of the pore opening. Pore sizes may bedetermined using techniques such as visible light microscopy, scanningelectron microscopy (SEM), and filtration methods, as described in moredetail below.

The cross-sectional shape (circular, oval, triangular, irregular, squareor rectangular, or the like), number, and dimensions of the pores can bevaried to suit a particular application. In one particular embodiment,the pores have an essentially circular cross-sectional profile. In somecases, the pores may have a smallest diameter that is smaller than asmallest cross-sectional dimension of a species to which the structuremay be exposed. These pores may, for example, prevent passage of thespecies across the pore, e.g., from a first side to a second side of aporous structure. In other cases, the pore size may be selected to bemuch larger than a species to which the structure may be exposed.Furthermore, in some instances, the spatial distribution of the poresmay be controlled.

In addition to the methods described above regarding fabrication ofpores by removal of a component from a structure precursor material,other methods of creating pores in a structure can also be used. In someembodiments, more than one technique for introducing porosity in astructure can be used. For instance, porosity can be induced in astructure by methods such as, for example, phase inversion, solutioncasting, emulsion casting, and polymer blending. For instance, pores canbe fabricated directly by a three-dimensional fabrication technique usedto fabricate the structure. E.g., arrays of holes or pores can be drawnonto a scanned image to form a porous skeleton of the imaged tissue ororgan. In other words, the pores can be fabricated using the samefabrication technique used to form the structure. In some cases, porescan be designed and printed with an offset. Additionally and/oralternatively, if desired, a porous material can be used to coat asurface of the structure. The porous material may include, for instance,more than one component having different solubility in certain solvents.For example, a first component may include the polymer in which thestructure is formed, and a second component may include particles thatare not soluble in the polymer, but which can be subsequently dissolvedin a solvent that dissolves the particles. After the structure is coatedwith the porous material, the structure can be soaked in a solvent thatdissolves the second component, e.g., to leach out the second componentfrom the porous material.

Accordingly, structures described herein may comprise pores having awide range of pore sizes. The pores of a structure may be uniform insize, or may vary in size if desired. In some embodiments, structuresdescribed herein are constructed to have a relatively homogeneous poresize distribution, for example, such that no more than about 5% of allpores deviate in size from the average pore size by more than about 20%,in some cases, by no more than about 10%, and in other cases, by no morethan about 5%. The pore size of the structure may be less than or equalto 1 mm, less than or equal to 100 microns, less than or equal to 50microns, less than or equal to 40 microns, less than or equal to 30microns, less than or equal to 10 microns, less than or equal to 5microns, less than or equal to 1 micron, or less than or equal to 100nm. In embodiments including more than one pore size, a combination ofpore sizes such as those described above, can be included in astructure.

As described herein, certain porous structures described herein may havesharp molecular weight cutoffs (MWCO). For example, at least 95% of thepores of a structure may have a MWCO of less than or equal to 5 kD, lessthan or equal to 10 kD, less than or equal to 15 kD, less than or equalto 20 kD, less than or equal to 25 kD, less than or equal to 30 kD,greater less or equal to 40 kD, less than or equal to 45 kD, less thanor equal to 50 kD, less than or equal to 55 kD, less than or equal to 60kD, less than or equal to 65 kD, less than or equal to 70 kD, less thanor equal to 75 kD, less than or equal to 80 kD, or less than or equal to100 kD.

Certain porous structures described herein may be able to excludecomponents having various sizes. For example, at least 95% of the poresof a structure may be able to exclude components having a size ofgreater than or equal to 1 mm, greater than or equal to 100 microns,greater than or equal to 50 microns, greater than or equal to 40microns, greater than or equal to 30 microns, greater than or equal to10 microns, greater than or equal to 5 microns, greater than or equal to1 micron, or greater than or equal to 100 nm.

In some embodiments, porous polymeric structures can be prepared in-situwith inherent properties that are suitable for use as biohybrid organsscaffolding. In some cases, structure precursor materials can be castedas flat sheet separation membranes and/or hollow fibers. The materialsmay have permeation properties ranging from the ultrafiltration tomicrofiltration ranges. These properties can allow the membranes toseparate substances having different molecular weights. In certainembodiments, the membranes can serve as bioactive membranes withoutfurther processing (e.g., further modification of the membrane surface),for example, the membranes may show good biocompatibility and celladherence without extracellular matrix (ECM) surface coatings.

In certain embodiments, structures such as flat membranes or hollowfibers can be fabricated using polysulfone (PS, a precipitatingcomponent) and Fullcure™ 700 monomer (FC, a crosslinkable component).The membranes and/or hollow fibers can be prepared with a controllableMWCO of between, for example, 5-100 kDa, which may allow the transportof certain ions, nutrients, waste products, protein-bound toxins, etc.The structures may be functionalized and modified by wet and dry surfacechemistry. In some cases, biospecific ligands can be covalently bound oradsorbed to the surfaces to support the attachment and function ofkidney epithelial cells on one side of the structure, and to achieve agood hemocompatibility on the blood-contacting side of the structure. Ifboth properties cannot be combined in one structure, replacement can beachieved by the application of a specific fiber-in-fiber design forhollow fibers.

FIG. 3 shown an example of a process for fabricating porous structuresin the form of membranes. As shown in the embodiment illustrated in FIG.3, first component 54 (e.g., a solution of polyethersulfone) including asolvent (e.g., dimethylacetamide) and second component 56 (e.g.,Fullcure™ 700 monomer) may be mixed to form a structure precursormaterial. The structure precursor material 60 may be poured andsandwiched between two glass plates 62 separated by one or more spacers64 that controls the membrane thickness. The whole assembly can then besubjected to UV radiation, as indicated by arrows 66, which can causepolymerization and/or crosslinking of one component of the structureprecursor material (e.g., Fullcure™ 700). The structure precursormaterial may be removed from the assembly and then subjected to a phaseinversion process 70, whereby the structure precursor material is placedin a precipitation medium 72 (e.g., water) to allow precipitation of onecomponent of the structure precursor material (e.g., polyethersulfone),and/or removal of a component of the material (e.g., the solvent), togenerate porous structure 74. The membranes can be highly permeable andmay have attractive biofunctional characteristics (such as sharp MWCO),and may possess high filtration and diffusion fluxes, and have goodmechanical strength, biocompatibility and formability. Such structuresmay be used in applications involving, for example, waste watertreatment and/or purification.

In some instances, pore sizes and/or the MWCO of a membrane can bemeasured by a filtration setup, as shown in FIG. 4. As illustrated inthe embodiments of FIG. 4, reservoir 80 can pump a feed solution fromlower compartment 86 to upper compartment 88 through porous membrane 90.Permeate may be collected in the upper compartment. The pumping andpressure of the solution can be controlled by pumping system 82 andpressure system 84, respectively. Using such a system, the flux of thesolution through the membranes can be measured under steady-state flow.The passing of different solutions containing solutes of known molecularweights (and/or sizes) can cause changes in the flux through themembrane, and the different concentrations of feed solution and permeatesolution can be used to determined the pore size and/or molecular weightcutoff of the membrane, as described in more detail in the Examples.

In some embodiments, articles of the invention can be used asbiocompatible structures for tissue engineering and/or organreplacement. Such structures may be formed, for example, bythree-dimensional fabrication techniques. In some embodiments, thebiocompatible structures are scaffolds for cells that can be used astissue engineering templates and/or as artificial organs. The structuresmay be three-dimensional and can mimic the shapes and dimensions oftissues and/or organs, including the microarchitecture and porosities ofthe tissues and organs. For instance, certain embodiments of theinvention can be fabricated to include very small features (e.g., lessthan 20 microns), such as small pore sizes, small cavities, and/orstructures having thin walls. These features are particularlywell-suited for structures involving hollow and epithelial organs. Insome cases, a structure formed by three-dimensional fabricationcomprises a wall defining a cavity and a plurality of pores in at leasta portion of the wall. The pores may permeate the wall, at least atselected portions of the wall or all throughout the wall, and enableexchange of a component (e.g., a molecule and/or a cell) between aportion interior to the cavity and a portion exterior to the cavity. Forinstance, pores may allow delivery of molecules, cell migration, and/orgeneration of connective tissue between the structure and its hostenvironment. Advantageously, structures including pores of uniform sizecan be fabricated by methods described herein. For example, pores mayhave an average pore size of less than or equal to 20 microns, whereinno more than about 5% of all pores deviate in size from the average poresize of the plurality of pores by more than about 20%. Structures of theinvention can be implanted into a mammal, or alternatively and/oradditionally, can be used ex vivo as bioartificial assist devices.

In some cases, structures can be fabricated to include substructures.For instance, a large vessel may be fabricated to include small vesselswithin the large vessel. Surfaces of substructures may also be modified,i.e., in a fashion described above. For example, in one embodiment, awall of the large vessel may be modified with a first growth factor toinduce growth of a first type of cell on the wall of the large vessel,and a wall of the small vessel may be modified with a second growthfactor to induce growth of a second type of cell on the wall of thesmall vessel. Substructures may include pores that allow exchange of acomponent between an interior cavity portion of the substructure and aportion exterior to the substructure, i.e., between a cavity portion ofthe substructure and a cavity portion of a larger structure.

A wide variety of artificial tissues and organs can be fabricated asthree-dimensional structures using methods described herein. In someembodiments, the structures can be used as templates for cell growth,which may be applied towards tissue engineering and/or organreplacement. For structures to be used in vivo, cells and/or tissues maybe grown on a structure prior to the structure being implanted, oralternatively, the structure may be positioned directly into a mammaliansystem where the body's cells naturally infiltrate the structure.

In some particular embodiments, structures may be formed in the shape oforgans that include a cavity portion. For instance, structures includinga cavity portion may include hollow organs and/or epithelial organs suchas vessels, lung, liver, kidney, pancreas, gut, bladder, and ureter, asdescribed in more detail below. A cavity of a structure, as used herein,refers to a substantially enclosed space defined by a wall of thestructure, in which a plane can be positioned to intersect at least onepoint within the cavity and the structure, where it intersects theplane, completely surrounds that point. The cavity and can be closed oropen. For example, in one embodiment, a cavity may be defined by theinterior space within a tube of a blood vessel. In another embodiment, acavity may be defined by the hollow space inside a bladder. As such,cavities may have a variety of shapes and sizes. A space within a cavityis referred to as an interior cavity portion, and a space outside of thecavity is referred to as a portion exterior to the cavity. The cavitymay be filled with fluid, air, or other components. In some cases, acavity may be lined with one or more layers of cells or tissues. Thelayers of cells or tissues may form, for instance, membranes or walls ofthe tissue or organ. In some instances, the lining of a cavity cancomprise pores that allow exchange of a component between a portioninterior to the cavity and a portion exterior to the cavity, asdescribed in more detail below.

A cavity of a structure may vary in volume and may depend, in someinstances, on the tissue or organ in which the structure mimics. Thevolume of the cavity may be, for instance, less than 1 L, less than 500mL, less than 100 mL, less than 10 mL, less than 1 mL, less than 100microliters, less than 10 microliters, less than 1 microliter, less than100 nanoliters, or less than 10 nanoliters, where volume is measured aswithin that portion of the structure that is enclosed.

A wall of a structure defining a cavity portion can vary in thickness,and may also depend on the tissue or organ in which the structuremimics. In some cases, thick walls (e.g., greater than 500 micronsthick) may be suitable for certain structures (e.g., a bladder) thatmay, for example, require slow or relatively little exchange ofcomponents between portions interior and portions exterior to thecavity. Thin walls (e.g., less than 50 microns thick) may be applicableto some structures (e.g., alveoli) that may, for example, require quickexchange of components between portions interior and portions exteriorto the cavity. In certain embodiments, a wall of a structure can be lessthan 1 mm thick, less than 500 microns thick, less than 200 micronsthick, less than 100 microns thick, less than 50 microns thick, lessthan 30 microns thick, less than 10 microns thick, less than 5 micronsthick, or less than 1 micron thick.

In some instances, a cavity may be defined by an inner diameter of acertain distance. “Inner diameter”, as used herein, means the distancebetween any two opposed points of a surface, or surfaces, of a cavity.For example, the inner diameter of a blood vessel may be defined by thedistance between two opposing points of the inner wall of the vessel.Inner diameters may also be used to describe non-spherical andnon-tubular cavities. A cavity may have an inner diameter of, forexample, less than 10 cm, less than 1 cm, less than 1 mm, less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, less than 30 microns, less than 10 microns, less than 5microns, or less than 1 micron.

In some embodiments, a structure may include a cavity having more thanone portion, for example, a first and a second cavity portion may beinterconnected, which allows a substance to pass freely between thecavity portions. Additionally or alternatively, the structure mayinclude more than one cavities (e.g., in a case where the cavities arenot interconnected). For instance, in one embodiment, a cavity of astructure may include at least a first and a second portion, the firstportion of the cavity being defined by a first inner diameter and thesecond portion being defined by a second inner diameter. In anotherembodiment, a structure may include a first cavity having a first innerdiameter and a second cavity having a second inner diameter. The secondcavity may be defined, for instance, by that of a substructure. For theabove cases, the first and second inner diameters may be different; forexample, the ratio of the first inner diameter to the second innerdiameter can be greater than 1:1, greater than 2:1, greater than 5:1,greater than 10:1, greater than 20:1, greater than 50:1, greater than100:1, greater than 200:1, or greater than 500:1. Some structures, suchas certain vessels, may have a first cavity portion having the sameinner diameter as that of a second cavity portion, i.e., the ratio ofthe inner diameters of the first and second portions may be 1:1.Additional examples of such structures are described in more detailbelow.

In mimicking tissues and/or organs of the body, different types of cellscan be arranged proximate a structure in sophisticatedmicro-architectures that are responsible for the complex functions ofthe tissue or organ. Thus, microstructures having dimensions andarrangements closely related to the natural conditions of the tissue ororgan can be formed. The design of the structure and the arrangement ofcells within the structure can allow functional interplay betweenrelevant cells, e.g., between cells cultured on the structure and thoseof the host environment. These factors may also enable appropriate hostresponses, e.g., lack of blood clotting, resistance to bacterialcolonization, and normal healing, when implanted into a mammaliansystem.

The present inventors have realized the importance of addressinggeometry, size, mechanical properties, and bioresponses in fabricatingstructures for tissue engineering and organ replacement, especially forstructures involving hollow and epithelial organs, as described in moredetail below.

In one aspect of the invention, tissues and organs of interest includethose of the circulatory system. The circulatory system includes theheart (coronary circulation), the blood vessel system (systemiccirculation), and the lungs (pulmonary circulation). The circulatorysystem functions to deliver oxygen, nutrient molecules, and hormones tothe body, and to remove carbon dioxide, ammonia and other metabolicwaste from parts of the body.

Coronary circulation refers to the movement of blood through the tissuesof the heart. In some cases, portions of the heart become diseased. Forinstance, heart tissue may not receive a normal supply of food and/oroxygen, or certain structures forming the heart, such as heart valves,may not be operating normally. In the latter case, when heart valves arefunctioning properly, the flaps (also called leaflets or cusps) of thevalves open and close fully. Proper function of heart valves may ceasewhen the valves do not open enough or do not let enough blood flowthrough; this condition is called stenosis. When the valves do not closeproperly, blood may leak into places where it shouldn't; this conditionis called incompetence or regurgitation. In these instances, heartvalves may need to be replaced. In one embodiment, methods describedherein can be used to fabricate heart valves (e.g., tricuspid,pulmonary, mitral, and/or aortic valves) that are coated with films ofadditives known to prevent blood clotting. In another embodiment, anartificial valve may incorporate additives such as antibiotics, whichcan prevent endocarditis, an infection of the heart's lining or valves.In some cases, an artificial valve may comprise a combination ofadditives, such as the ones mentioned above. The heart valves can beused in vivo to replace diseased heart valves, and/or in vitro as ascaffold template for cell seeding.

In another embodiment, three-dimensional fabrication techniques can beused to form structures of the blood vessel system, including arteries,veins, capillaries, and lymphatic vessels. The blood vessel system keepsblood moving around the body inside the circulatory system.

Arteries carry blood that is full of oxygen from the heart to all partsof the body. As the arteries get further away from the heart, they getsmaller. Eventually, arteries turn into capillaries, the smallest bloodvessels, which go right into the tissues. Here, the blood in thecapillaries gives oxygen to the cells and picks up the waste gas, carbondioxide, from the cells. The capillaries are connected to the venules,the smallest veins in the body, and the veins get bigger as they carrythe blood back towards the heart. The capillaries are the points ofexchange between the blood and surrounding tissues. Components can crossin and out of the capillaries, for instance, by passing through orbetween the cells that line the capillary.

Structures for use as templates for cell growth can be designed to mimica variety structures of the blood vessel system. In some embodiments,structures can serve as templates for triggering controlled in-growth ofvascular structures or complete artificial vessel replacements. Suchstructures may be used for the induction of vessels in vivo.

Structures described herein may be formed in the shape of a tubeincluding interior cavity portion and a portion exterior to the cavity.The structure may have a first end portion and a second end portion,which may be opened or closed. In some cases, the end portions and maybe used to connect the structure to ducts of a patient. The dimensionsof the structure may vary depending on the particular body part thestructure will mimic, where the structure will be positioned in thebody, the size of the patient, etc. For example, the structure may havean inner diameter and/or outer diameter of less than 10 mm, less than 5mm, less than 2.5 mm, less than 1.5 mm, or less than 1 mm. In someembodiments, the structure can be transplanted into a mammalian body andmay have a length between 10 mm and 100 mm, or between 25 mm and 75 mm(e.g., 50 mm); the inner diameter may have a length of about 0.5 mm, andthe outer diameter may have a length of about 1.5 mm. The thickness ofthe wall of the structure may be defined by the difference between innerand outer diameters. Thicknesses of the wall can range from a fewmicrons (i.e., a few cells) to millimeters thick.

In some cases, the structure can have a plurality of pores in at least aportion of the structure. The pores can vary in size; for instance,large pores (e.g., greater than 100 microns) may be suitable for growinglarge vessels through the pores, and/or for facilitating high exchangeof components between an interior cavity portion and portion exterior tocavity. Small pores (e.g., less than 100 microns) may be suitable forgrowing small vessels through the pores, and/or for facilitatingrelatively low exchange of components across the wall of the structure.Such structures may be implanted into a mammal, or used in vitro.

In some cases, structures may include one or more additionalsubstructures. For instance, a tubule may be fabricated to include asubstructure such as a vessel. The vessel may be positioned in at leasta portion within an interior cavity portion of the tubule, or it may bepositioned exterior to the cavity. In some cases, the vessel may passacross a pore of the tubule, or the vessel may be interwoven betweenpores of the tubule. As such, the tubule may include at least a firstcavity (e.g., an interior cavity portion of the tubule) and a secondcavity (e.g., a cavity portion of the vessel). The ratio of the innerdiameter of the first cavity to the inner diameter of the second cavitymay be, for example, greater than 1:1, greater than 2:1, greater than5:1, greater than 10:1, greater than 20:1, greater than 50:1, greaterthan 100:1, greater than 200:1, or greater than 500:1.

In some embodiments, structures described herein can be used to replacea section of a blood vessel in a patient. Such a structure may includean interior cavity portion having an inner diameter, a portion exteriorto the cavity, a first end, and a second end. The structure can alsoinclude sections that can be used as interconnecting lumens forconnecting the structure to one or more ducts of a patient. If desired,the structure can be designed to include a plurality of such sections.The sections may each be defined by cavity portions having a certaininner diameter. In some cases, the ratio of the inner diameter of afirst cavity portion to the inner diameter of a second cavity portioncan be equal to 1:1, greater than 1:1, greater than 2:1, greater than5:1, greater than 10:1, greater than 20:1, greater than 50:1, or greaterthan 100:1.

The wall of the structure may have a thickness of less than 5 mm, lessthan 1 mm (e.g., 0.5 mm), or less than 0.5 mm. In one particularembodiment, a wall of a structure has a thickness of 0.5 mm. In somecases, the wall may be formed in an elastic material that allowsstretching, recoiling, and/or absorption of pressure in response to, forexample, pumping of the heart and fluid flow through the structure. Ifdesired, before implanting the structure into a patient, smooth musclecells may be grown onto all, or portions, of the wall of the structure.These muscle cells may contract and expand to control the diameter, andthus the rate of blood flow, through the structure (e.g., contractionand expansion of muscle cell may cause the structure to dilate andconstrict, respectively). In some cases, an additional outer layer ofconnective tissue may be grown onto the structure. A layer of elasticfibers may also be grown onto the structure to give it greaterelasticity, if desired. In some embodiments, the structure can be madefrom a biodegradable polymer that degrades, for example, after healthytissues have re-grown and have integrated into the body.

In some embodiments, structures formed by methods described herein aredesigned to mimic capillaries, which can allow exchange of componentssuch as nutrients, wastes, hormones, and white blood cells, between theblood and surrounding environment. The surrounding environment mayinclude, for example, the interstitial fluid and/or surrounding tissues.The artificial structure may include a cavity portion comprising a wallhaving a thickness of, for example, 0.5 mm or any other suitablethickness, which can be lined with endothelial cells. In some cases, awall of the capillary has a thickness of a single cell. In oneembodiment, capillary structures may include small pores or holes thatmay be less than 50 microns, less than 10 microns (e.g., about 1 micron)in size between the cells of the capillary wall, allowing certaincomponents to pass in and out of capillaries, e.g., between an interiorcavity portion and a portion exterior to the cavity (e.g., thesurrounding tissues). The pores may allow certain small components suchas certain dissolved molecules (e.g., small ions) to pass across thepores, but may inhibit larger components such as proteins from passingacross. In another embodiment, exchange of components across a capillarywall can occur by vesicles in the cells of the capillary wall that pickup components from the blood (e.g., in the interior cavity portion ofthe capillary), transport them across the capillary walls, and expelthem into the surrounding tissue (e.g., into a portion exterior to thecavity of the capillary). In yet other embodiment, components mayexchange between an interior cavity portion and a portion exterior tothe cavity via passage through the cell lining. For instance, componentsmay diffuse from the blood into the cells of the capillary walls, andthen into the surrounding tissue. Artificial capillaries may also bedesigned to include one or more branching structures, which can create agreater surface area through which the exchange of components can occur.

In another aspect of the invention, structures are fabricated to mimictissues and/or organs of the digestive track. The digestive tractencompasses the oral cavity, esophagus, stomach, small and largeintestines, rectum, and anus. The different parts of the digestive tractmay display a similar histo-architecture, i.e., each part may comprise amuscle wall that is covered by the mucosa, which contains epithelialcells. These organs can be affected by diseases such as cancer,infection, etc. Diseased organs of the digestive track typically requireoperations that include resections of the diseased segment. Theseremoved segments can be replaced with artificial structures of thepresent invention. In some embodiments, structures can be fabricated tomimic a diseased section. The structure may be used as a scaffold forthe in-growing of natural mucosa from healthy cells of a patient. Thisscaffold can then be implanted into the patient. In one embodiment, thisapproach is applied to so-called gut pouches to replace the continencefunction of the gut. Like artificial structures of the circulatorysystem, structures of the digestive track can be formed in biodegradablepolymers.

In another aspect of the invention, structures are fabricated to mimicgut-associated glands. Gut-associated glands include the salivaryglands, the liver, and the pancreas. All three organs are made up ofspecialized epithelial cells with endocrine and exocrine functions. Inone embodiment, structures can be fabricated to mimic portions of theliver. The liver is comprised mainly of lobules containing hepatocytesthat are arranged in plates. In between the hepatocyte plates,blood-containing sinusoids can be found. The center of the lobule is thecentral vein, and this vessel receives blood from the sinusoids. In someembodiments, artificial structures in the shape of liver lobules can befabricated. The structure may include a scaffold for plating and growinghepatocytes. The scaffold can be designed with a specificmicro-architecture that allows spatial control of the seeding of cells.The structure may also include sinusoidal structures, which can functionas cavities for containing blood. The plates can be filled withhepatocytes in the inner space of the scaffold, and a plate walladjacent a center can be coated with endothelial cells. In certainembodiments, the liver lobules can have dimensions of approximately 0.7mm×2 mm. The structure can be fabricated to have pores that canfacilitate exchange of a component. For example, exchange of a componentmay occur via pores between the blood contained in the sinusoidalstructures (e.g., an interior cavity portion) and the hepatocytes, whichmay be located at a portion exterior to the cavity. Pores can befabricated to have a variety of sizes. Generally, for liver lobules,pores may be fabricated to have a cross-sectional dimension in themicron range.

In another embodiment, structures for tissue engineering and/or organreplacement can be fabricated to mimic portions of the pancreas. Thepancreas is a mixed exocrine-endocrine gland that produces hormones suchas insulin and glucagons, as well as pancreatic enzymes that help digestacids and macromolecular nutrients (e.g., proteins, fats and starch).The hormone-producing cells are aggregated in the islets of Langerhans.Pancreatic islets are scattered throughout the pancreas. Like allendocrine glands, pancreatic islets secrete their hormones into thebloodstream and not into tubes or ducts. Because of the need to secretetheir hormones into the blood stream, pancreatic islets are surroundedby small blood vessels (e.g., capillaries). The islets are also highlyvascularized, facilitating the exchange of hormones between the isletsand the vessel system. In certain embodiments of the invention,structures in the shape of island-like structures are fabricated usingtechniques described herein. The artificial island-like structures canbe designed to have a specific micro-architecture that can enableendocrine cells to be seeded in preformed locations, i.e., nearstructures that are designed to guide the capillaries. Like thestructures described above, structures that mimic portions of thepancreas can be formed in biodegradable polymers if desired. Theseartificial pancreatic structures may be used to treat diseases such asdiabetes mellitus.

In another aspect of the invention, structures are fabricated to mimicendocrine organs. The endocrine organs include the adrenals, thyroid,parathyroid, and pineal gland. These organs are made up of endocrine(i.e., hormone-producing) cells that are located very close to thecapillaries, as described above for the islets of Langerhans. The closeproximity of these organs to the capillaries allows the bloodcirculating factors to leave the capillaries and become bound to cellreceptors on the endocrine cells, triggering the release of hormones.The released hormones diffuse into the capillaries, and are subsequentlydistributed in the body to bind with receptors in other tissues. In someembodiments, endocrine structures can be fabricated to have a specificmicro-architecture that allows the seeding of cells within certainlocations of the structure. Artificial endocrine organs may befabricated to have a high degree of vascularization that facilitates theexchange of components between the organ and the capillaries. In somecases, artificial endocrine organs are made with high porosity. Thepores may have a variety of sizes depending on the particular organ.Like the structures described above, structures that mimic endocrineorgans can be formed in biodegradable polymers if desired. Artificialendocrine organs may be applied, for instance, towards treatinginsufficient production of hormones in glands.

In another aspect of the invention, structures are fabricated to mimicportions of the respiratory system. The respiratory system includes thetrachea and the lungs. In one embodiment, a structure can be fabricatedto replace diseased or damaged portions of the trachea. The trachea is acartilaginous and membranous-ringed tube where air passes to the lungsfrom the nose and mouth. The trachea bifurcates into right and leftmainstem bronchi. Artificial trachea may be fabricated to includesimilar architecture and mechanical properties to that of healthytrachea. For instance, the artificial structure may include ring-likeportions made from an elastic polymer that resembles cartilage. In somecases, cartilage cells (e.g., hyaline cartilage) from healthy tracheacan be seeded and grown into the artificial structure. The artificialstructures can be lined with ciliated cells, used to remove foreignmatter (e.g., dust) from the airway so that they stay out of the lungs.

In one embodiment, a structure can be fabricated to replace diseased ordamaged portions of the lung. The lungs include air-conducting segmentssuch as the bronchioles, numerous small tubes that branch from eachbronchus (a branch of the trachea) into the lungs. The lungs alsoinclude the alveoli, the respiratory portions where gas exchange takesplace. The air-conducting portions include a wall that is lined byrespiratory epithelium, which is responsible for producing mucous fluid.In some cases, structures are fabricated to mimic portions of theair-conducting segments. For instance, artificial bronchioles may befabricated to have a thickness of less than 10 mm, less than 1.0 mm(e.g., 0.5 mm), or less than 0.5 mm, and a diameter of less than about10 mm, less than about 5 mm (e.g., 2 mm), or less than about 2 mm. Thethickness and diameter of the bronchiolar structure will depend, ofcourse, on the position of the structure within the lung, the size ofthe patient, etc. All structures of the air-conducting portion can beformed as the artificial interposed segments or as templates forengineered tissue constructs. For instance, in some cases, theartificial structure may form a scaffold for growing connective tissueand smooth muscle cells within the walls of the structure. The walls mayalso be lined with epithelial cells, which can comprise three types ofcells: ciliated cells, non-ciliated cells, and basal cells. In someparticular embodiments, certain artificial structures, such as thosethat mimic terminal bronchioles, can be fabricated to include artificialalveoli in the walls of the structure.

In some embodiments, structures are fabricated to mimic alveoli. Alveoliare small, thin-walled air sacs (i.e., cavities) at the end of thebronchiole branches having cross-sectional dimensions on the order of200 microns. Proximate the alveolar walls are pulmonary capillarieswhere gas exchange occurs between blood in the capillaries and inhaledair in the alveoli. For instance, to reach the blood, oxygen diffusesthrough the alveolar epithelium, a thin interstitial space, and thecapillary endothelium; carbon dioxide follows the reverse course toreach the alveoli. In certain embodiments of the invention, artificialalveolar structures can be fabricated with natural dimensions and withporous walls for gas exchange. Pores in the walls of the alveoli mayallow exchange of a component (e.g., a gas) between an interior portionof the alveoli (e.g., an interior cavity portion) and the interstitialspace surrounding the alveoli (e.g., a portion exterior to the cavityportion). Artificial alveolar structures may be formed in an elasticmaterial that gives the alveoli mechanical stability while allowingexpansion and contraction of the structures. In some cases, theartificial alveolar structures may form scaffolds for growing cells,i.e., the structures may be lined with epithelial cells such as Type 1and Type 2 pneumocytes. Artificial alveoli can be used to help increasethe oxygen content in patients with respiratory deficiencies.

In another aspect of the invention, structures are fabricated to mimicportions of urinary system. The urinary system comprises the kidneys,ureters, the urinary bladder, and the urethra. In some cases, astructure can be fabricated to replace diseased or damaged portions ofthe kidney. The kidney is formed from a plurality of nephrons, whichinclude the glomerulus and the proximal and distal convoluted tubules.The glomerulus represent the filtration stations, which contain tuffs ofcapillaries where the ultrafiltrate is pressed out. In some embodiments,a structure can be fabricated in the form of a porous loopedsuperstructure. In one embodiment, the structure can be used as anartificial glomerulus. In another embodiment, the structure can be usedas artificial proximal and/or distal convoluted tubules. In some cases,the structure can include a plurality of loops, which can be of the sameor different dimensions. The structure can include at least one walldefining a cavity (e.g., a tubular portion). The cavity can have thesame inner diameter throughout the structure, e.g., of about 40-500microns in one embodiment, or between 50-100 microns in anotherembodiment. Alternatively, a first portion of the cavity may have aninner diameter different than that of a second portion of the cavity.The thickness of the wall can range, for example, from about 1-500microns (e.g., 2-500 microns), 1-100 microns, or 2-100 microns. The wallmay optionally include a plurality of pores that enable exchange of acomponent (e.g., water and ions) between a portion interior to thecavity and a portion exterior to the cavity. The pores may allow certaincomponents to pass between interior and exterior portions of the cavity,e.g., based on size, charge, etc. In some cases, all, or portions, ofthe wall can be covered with films of nanometer to micron thickness.These films can form selective permeable membranes allowing certaincomponents to pass between interior and exterior portions of the cavity.The structure may be used to process ultrafiltrate in such a way thatthe good substances (e.g., glucose and amino acids) become reabsorbed,and the wastes (e.g., urea) get discarded as urine. In certainembodiments, the structure can act as a hemofiltration system.Accordingly, the structure can be used to replace and/or aid thefiltration function of the kidney.

Some embodiments of the invention include the formation of a pluralityof cavities within a structure. For example, in the embodiment, astructure can be formed in the shape of a block, and may include a wallthat defines a plurality of cavities. Cavities within the structure maybe separate in some embodiments, or they may be interconnected in otherembodiments. Cavities within the structure may have the same ordifferent geometry and/or dimensions. Structures having a plurality ofcavities can be used, for instance, to improve the surface-to-volumeratio in a hemofiltration system, e.g., for higher rate of reabsorptionof electrolytes such as glucose and other metabolic products. In somecases, such structures may be combined with other embodiments of theinvention. For instance, such structures can be combined with one ormore artificial glomeruli to replace the main renal function with anextracorporeal module. In other instances, such structures can becombined with one or more artificial glomeruli as an implantable deviceto replace the main renal function in a mammalian system.

In some cases, structures including a plurality of cavities may includeone or more additional substructures. For instance, such a structure maybe fabricated to include a substructure such as a vessel. Thesubstructure may be positioned in at least a portion of a cavity of thestructure, or the substructure may be positioned exterior to the cavity.In some cases, a substructure may be interwoven between more than onecavities of the structure. As such, the structure may include at least afirst cavity and a second cavity (e.g., a cavity portion of the vessel).The ratio of the inner diameter of the first cavity to the innerdiameter of the second cavity may be, for example, greater than 1:1,greater than 2:1, greater than 5:1, greater than 10:1, greater than20:1, greater than 50:1, greater than 100:1, greater than 200:1, orgreater than 500:1.

In some cases, an artificial structure can be fabricated to replacediseased or damaged portions of the ureter and/or bladder. The ureterand bladder are hollow organs that include a wall, lined by atransitional epithelium, defining a cavity portion. Sometimes, thisepithelium can be affected by cancer. Typically, to treat such adisease, a surgical operation is necessary whereby portions of the gutare removed and used to replace the reservoir function of the bladder,or the conductive function of the ureters. In some cases, this procedurecauses the urethra to be affected by infection, leading to urethrastenosis. To circumvent these complications, diseased portions of theureter and/or bladder may be replaced using artificial structures of theinvention. Artificial structures may also be used to replace portions ofthe ureter and/or bladder to treat conditions such as urinaryincontinence.

Structures formed by three-dimensional fabrication techniques can beused to replace portions of the ureters or urethra, or, they may beemployed as artificial urinary bladders. The structures may be used fortissue engineering and/or organ replacement, in vivo or ex vivo. In oneembodiment, a structure to be used as an artificial bladder includes amain body portion, an inlet for connecting to the ureters, and an outletfor connecting to the urethra. The structure may include a wall defininga cavity portion of the main body portion (e.g., a first cavityportion), a cavity portion of the inlet (e.g., a second cavity portion),and a cavity portion of the outlet (e.g., a third cavity portion). Thecavity portions may have inner diameters ranging from, for example,about 0.01-5 mm, or 0.01-2 mm. In some instances, one cavity portion mayhave an inner diameter that is different from the inner diameter ofanother cavity portion of the structure. For example, the ratio betweeninner diameters of the second cavity portion and the first cavityportion may be greater than 1:1, greater than 2:1, greater than 5:1,greater than 10:1, greater than 20:1, greater than 50:1, or greater than100:1.

The wall of the structure to be used as an artificial bladder may have athickness ranging from, for example, about 0.01-5 mm, or 0.01-2 mm,depending on the volume of liquid in the artificial bladder, and may beformed in a flexible material to allow expansion and contraction of thebladder. In some cases, the wall is lined with cells and/or tissuesbefore implanting the structure into a patient. For instance, thestructure may serve as a template for different layers of tissues thatform the bladder, e.g., the mucosa, submucosa, and muscularis layers.The mucosa includes the transitional epithelium layer, which can serveas a selective barrier between the organ an environment exterior to theorgan. Underneath the epithelium layer can include the basementmembrane, a single layer of cells separating the epithelial layer fromthe submucous layer (lamina propria). The submucous layer includesconnective tissue that is interlaced with the muscular coat. Thesubmucous layer can contain blood vessels, nerves, and in some regions,glands; in some embodiments, the structure can include suchmicro-architectures. Muscle cells defining the muscular layer may bepositioned underneath the submucous layer.

Structure described herein may be capable of or modified to permit theadhering of various species to the surface of the structure or to amaterial coating a surface of the structure. For example, cells and/orbiological molecules such as proteins, and the like may becomeimmobilized with respect to various portions of the structure,including, for example, areas along the side walls of the pores, areasbetween the pores on a surface of the structure, or areas on top of thepores.

Some structures described herein may comprise an adhesive materialselected to preferentially attract and/or bind a particular species,such as a cell or other biological species that is attached to,immobilized with respect to, or otherwise associated with at least oneside of a structure. In certain embodiments, the adhesive material is acell adhesive material. The term “cell adhesive material” as used hereinmay refer to any chemical or biological material to which a cell mayadhere. In certain embodiments, such a cell adhesive material isconfigured as a continuous layer attached to a surface of at least oneside of a structure. Such a cell adhesive material layer may comprise,any of a wide variety of species known in the art to be capable ofbinding to, specifically or non-specifically, membranes of biologicalcells or components thereof, such as for example, collagen or mixturesof collagen with polysaccharide, antibodies, ligands to cell surfacereceptors, antigens, lectins, integrins, selectins, bacterial derivedaffinity molecules such as Protein A or Protein G, derivatives thereof,mixtures thereof, any of the above associated with a gel or otherlayer-forming material, such as collagen, gelatin, agarose, acrylamide,chitosan, cellulose, dextran, an alginate, a carrageenan, etc., and thelike.

Surface properties of the structures can be modified by varioustechniques. In some cases, surfaces of a structure can be modified bycoating and/or printing an additive proximate the structure. In othercases, additives can be incorporated into the material used to form thestructure (e.g., embedded in the structure during fabrication), asdescribed herein. Surfaces may be modified with additives such asproteins and/or other suitable surface-modifying substances. Forexample, collagen, fibronectin, an RGD peptide, and/or otherextracellular matrix (ECM) proteins or growth factors can be coated ontothe structure, e.g., to elicit an appropriate biological response fromcells, including cell attachment, migration, proliferation,differentiation, and gene expression. Cells can then be seeded ontosurfaces of this structure. In one embodiment, cell adhesion proteinscan be incorporated into certain channels and/or pores of a structure tofacilitate ingrowth of blood vessels in these channels and/or pores. Inanother embodiment, growth factors can be incorporated into thestructure to induce optimal cell growth conditions that triggers healthytissue formation within certain regions of the structure. In yet anotherembodiment, a structure may comprise an additive such as a cell adhesivematerial positioned on one surface of a side of the structure, such asan inner cavity of a structure. In such an embodiment, a first type ofcells can adhere to the inner cavity of the structure when the structureis exposed to a medium containing cells. Optionally, an outer portion ofthe structure may preferentially attract and/or bind a second type ofcell when the structure is exposed to a medium containing cells.

In some cases, it may be desirable to modify all or portions of asurface with a material that inhibits cell adhesion, such as asurfactant (e.g., polyethylene glycol and polypropyleneoxide-polyethylene oxide block copolymers). For instance, areas of astructure where it is not desirable for cellular growth can be coatedwith such materials, e.g., to prevent excessive soft connective tissueingrowth into the structure from the surrounding tissue. In some cases,modification of surface properties of the structure can be used toposition cells at specific sites within the structure. In someembodiments, a combination of cell-adhering and cell-inhibitingsubstances can be incorporated into various portions of a structure tosimultaneously facilitate and inhibit cell growth, respectively.

In some embodiments, a structure can be coated with a porous material(e.g., a polymer such as a gel) prior to being coated and/or printedwith a surface-modifying substance. For instance, in one embodiment, astructure can be fabricated using three-dimensional fabrication oranother suitable technique to form a bioartificial kidney. In someinstances, the structure can be modified with a substance; for instance,the structure can be first coated with a porous polymer, and then with asurface-modifying substance such as collagen, which may be used tofacilitate cell adhesion. Cells (e.g., vascular cells) can then beseeded into and/or onto the modified structure. In some cases, thestructure may include another layer of cells (e.g., proximal tubulecells). The device may mimic the function of a kidney to allow flow ofblood and ultra-filtrate in and out of the structure.

If desired, structures of the invention can be coated with a porouspolymer. A porous polymer coating a structure can be used for a varietyof purposes. For example, a porous polymer may be used to form smallpores (e.g., having a cross sectional dimension on the order of 1-20microns, or within the range of porosity of the polymer) within a largerpore (e.g., having a cross sectional dimension on the order of 20-200microns) of the structure. In some cases, the porous polymer may allowsustained release of an active agent from the polymer, e.g., tofacilitate cell growth and/or adhesion as a function of time. In othercases, the porous polymer can influence transport of components from afirst to a second position of the structure. In yet other cases, aporous polymer coating a structure can reduce the surface roughness ofthe structure, as described below. One non-limiting example of asuitable porous polymer is polysulfone.

A variety of techniques, such as those described below, can be used tofabricate or shape structures described herein. After or during theprocess of carrying out such techniques, the structure may be exposed toa precipitation medium to form cell growth template structures having anetwork uniform pores.

In some embodiments, structures described herein are fabricated at leastin part by using one or more ejection processes, such as jettingprocesses, including thermal and/or piezo jetting, such as by use of anink jet device, for example. In one particular embodiment, a printingtechnique using a printer is used to fabricate a three-dimensionalstructure from thin, two-dimensional (“2D”) layers. A computer is usedto generate cross-sectional patterns of the 2-D layers by storing adigital representation of the object in a computer memory. Acomputer-aided design or computer-aided manufacture (“CAM”) software isthen used to section the digital representation of the structure intomultiple, separate 2D layers. A printer, such as an inkjet printer, isused to fabricate a layer of structure precursor material for each layersectioned by the software, onto a flat surface or support platform,optionally using a roller. The structure precursor material may be inthe form of a liquid or a powder and may be, for example, a ceramic,metal, polymeric, or composite material. If the structure precursormaterial is in the form of a powder, a liquid binder is selectivelydeposited on the powder material using a printhead of the inkjet printerto produce areas of bound powder. The liquid binder, which is typicallya polymeric resin or aqueous composition, is applied in the pattern ofthe cross-sectional pattern of the 2D layer. The liquid binder canpenetrate gaps in the powder material and may react with the powderparticles to create a layer bound in two dimensions. As the reactionproceeds, the binder also bonds each successive 2D layer to a previouslydeposited 2D layer. Additional 2D layers are formed by repeating thesteps of depositing additional structure precursor material and applyingthe binder solution until the desired number of layers is produced.Since the liquid binder is selectively applied to the powder material,only certain areas of the powder material are bound within the layer andonto the previous layer. After the 3D object is formed, unbound powderis subsequently removed, e.g., by dissolving the powder in anappropriate solvent. The precursor structure may then be polymerizedand/or crosslinked, and/or exposed to a precipitation medium to form thefinal porous structure.

In some embodiments, structures described herein are formed at least inpart using a multi-photon lithography system. For instance, two-photonlithography or three-photon lithography systems may be used.Multi-photon polymerization may involve the use of an ultra-fastinfrared laser (e.g., a femtosecond laser operating at a wavelength of1028 nm), which can be focused into the volume of a structure precursormaterial including a photosensitive material. The polymerization processcan be initiated by non-linear absorption within the focal volume. Bymoving the focused laser three-dimensionally through the resin,three-dimensional structures can be fabricated.

In one embodiment, a two-photon lithography system can be used at leastin part to fabricate structures for tissue engineering and/or organreplacement. In a two-photon lithography system, a monomer mixed with aphoto initiator that absorbs UV light may be exposed to an infra-redlaser. Two photons of infra red light can be absorbed by theresin/chemicals and a single photon of ultra-violet light can bereleased. The released photon can then be absorbed by the photoinitiator to produce free radicals which can cause polymerization of themonomers. Since the two-photon absorption cross-section is very small,for the release of sufficient UV light to induce free radicalpolymerization in the chemicals, a large amount of energy (terawatt) canbe delivered to the chemical by the laser. This energy density could begenerated at the focal point of a laser beam from an ultra-fast (e.g.,femtosecond) pulse laser. Two-photon-absorption only occurs at the focalpoint of the beam and not at the laser beam path, hence a very smallvolume (e.g., femtoliter) of monomer can be polymerized through therelease of free radicals from the photo initiator. After the structurehas been polymerized, e.g., from a block of resin or in a petri dish ofmonomer, the unexposed chemicals can be washed away with a suitablesolvent, leaving behind the final structure. The technique can been usedwith a variety of materials, including acrylate and epoxy polymers suchas ethoxylated trimethylolpropane triacrylate ester and alkoxylatedtrifunctional acrylate ester, as described herein. This system can beused, for instance, when structures with fine resolution are desired.E.g., in some cases, multi-photon lithography can be used to formstructures having submicron (e.g., less than one micron) resolution.

In one embodiment, stereolithography can be used at least in part toform structures for tissue engineering and/or organ replacement.Stereolithography may involve the use of a focused ultra-violet laserscanned over the top of a reservoir containing a photopolymerizableliquid polymer. The UV laser can cause the polymer to polymerize and/orcrosslink where the laser beam strikes the surface of the reservoir,resulting in the formation of a solid or semi-solid polymer layer at thesurface of the liquid. The solid layer can be lowered into the reservoirand the process can repeated for formation of the next layer, until aplurality of superimposed layers of the desired structure is obtained.This process may allow formation of various self-supporting structures,which may then be exposed to a precipitation medium to form cell growthtemplate structures having a network uniform pores.

In another embodiment, selective laser sintering (or laser ablation) canbe used at least in part to form structures for tissue engineeringand/or organ replacement. Selective laser sintering may involve the useof a focused laser beam to sinter areas of a loosely-compacted plasticpowder, where the powder is applied layer by layer. For instance, a thinlayer of powder can be spread evenly onto a flat surface, e.g., using aroller mechanism. The powder can be raster-scanned using a high-powerlaser beam. The areas of the powder material where the laser beam wasfocused can be fused, while the other areas of powder can remaindissociated. Successive layers of powder can be deposited andraster-scanned, one on top of another, until an desired structure isobtained. In this process, each layer can be sintered deeply enough tobond it to the preceding layer.

In some embodiments involving three-dimensional fabrication, variationof the laser intensity and/or traversal speed can be used to vary thecrosslinking density within a structure. In some cases, this allows theproperties of the material to be varied from position to position withthe structure. Variation of the laser intensity and/or traversal speedcan also control the degree of local densification within the material.For instance, regions where the laser intensity is high or the traversalspeed is low can create areas of higher density.

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

Example 1 Preparation of Polysulfone-Fullcure™ (PS-FC) Membranes

PS-FC membranes were prepared from polysulfone (PS, Sigma Aldrich,MW=26,000 g/mol) and Fullcure™ 700 monomer (Stratasys, USA) in weightratios indicated in Table 1. The solvent, N,N-dimethylacetamide (DMAc,Sigma Aldrich), was used as received. FIG. 3 illustrates the synthesisscheme for the membrane preparation. A solution of PS in DMAc (10 wt %)and Fullcure™ 700 monomer was poured and sandwiched between two glassplates separated by a spacer that controlled the membrane thickness.Using this assembly, 80-micron-thick membranes were fabricated. Thewhole assembly was then subjected to UV curing for 30 min, which fixedthe spatial arrangement of the polymer blend to form a free-standingstructure. The structure was then immersed in a water bath to carry outa phase inversion process (a solvent-non-solvent treatment process,wherein water was used as the non-solvent (precipitation medium)) thatled to the precipitation of PS to generate a porous network. After themembrane peeling away from the glass plate, the membrane was washed withdistilled water and stored in distilled water at room temperature beforeuse.

TABLE 1 Composition and properties of PS-FC membranes. Blend CompositionPS-FC-0.15 PS-FC-0.2 PS-FC-0.25 PS (g) 2.0 2.0 2.0 FC (g) 0.15 0.20 0.25Pore Diameter^(a) (μm) 10-15 6-12 5-10 Pore Diameter^(b) (nm) 12.5 8.65.5 MWCO (kDa) 80 40 15 Pure Water Flux 717 597 161 (L/m² · h) T_(g) (°C.) 90, 195 95, 198 97, 200 Contact Angle (°) 30 35 38 Storage Modulus1550 1920 2100 Biocompatibility Yes Yes Yes ^(a)Measured by SEM.^(b)Measured by MWCO.

Example 2 Characterization of PS-FC Membranes

The PS-FC membranes of Example 1 were characterized by scanning electronmicroscopy (JEOL JSM-7400F, 10 kV). The separation properties of themembranes were examined using the solute rejection technique forultrafiltration membranes. The membranes were cut into the necessarysize for use in the ultrafiltration cell.

The membranes were subjected to a pressure of 20 psi, and the flux ofwater through the membranes was measured under steady-state flow usingEq. 1:

$\begin{matrix}{J_{w} = \frac{Q}{A\; \Delta \; t}} & (1)\end{matrix}$

where Q is the quantity of permeate collected (L), J_(w) is the waterflux (L/m²·h), ΔT is the sampling time (h), and A is the membrane area(m²).

The pore size of the PS-FC membranes was determined by theultrafiltration of polyethyleneglycol (PEG) with different molecularweights. A standard curve of the PEG solution was obtained using purePEG fractions varying from 2 to 100 kDa. The molar masses of PEG wereobtained by gel permeation chromatography. All the PEG solutions wereprepared at a concentration of 1 wt %, and used as the feed. Higherconcentrations were avoided since the permeate flux would decline withincreasing feed concentration and affect the rejection performance. TheMWCO values were calculated using Eq. 2:

$\begin{matrix}{{\% \mspace{14mu} S\; R} = {\left\lbrack {1 - \frac{C_{p}}{C_{f}}} \right\rbrack \times 100}} & (2)\end{matrix}$

where SR corresponds to 90% solute rejection and C_(f) and C_(p) are thefeed and permeate concentrations (mol/dm³), respectively. The averagepore radius r (Å) of the membrane was calculated from the MWCO value ofthe PEG by Eq. 3:

r=0.33(M)^(0.46)  (3)

where M is molecular weight of solute.

The properties of the PS-FC membranes are summarized in Table 1. PS-FCmembranes prepared with a PS:FC weight ratio smaller than 2:0.25possessed finer and less interconnected pores. The PS-FC membranesshowed two T_(g) values at ˜100 degrees Celsius and ˜200 degreesCelsius, and high storage modulus ranging from 1550 to 2100 MPa. Ahigher storage modulus was obtained for the membrane with a higher FCcontent, which provided a more elastic framework to the porous PS. Thethree membranes showed similar water Contact angles in the range of30-38 degrees, indicating that the membranes could provide moderatelywettable surfaces for the attachment and proliferation of tissue cells.

In all of the PEG rejection studies for determining the pore statisticsand MWCO, the feed side was uniformly agitated to prevent concentrationpolarization and cake formation on the membrane surface, which wouldaffect the flux, and ultimately, the partition coefficient and aggregatepore size.

PS-FC-0.15, PS-FC-0.20 and PS-FC-0.25 blend membranes were subjected towater flux assessment at a pressure of 20 psi, and compared to thecommercial BTS-45 and BTS-55 PS membranes (Pall Corporation, USA) having0.3 and 0.2 micron sized pores, respectively. As shown in FIG. 5, alower water flux was observed for PS-FC-0.25, compared to PS-FC-0.15 andPS-FC-0.20, which showed a steady-state flux of 717 and 597 L/m²h,respectively. The decrease hi flux with increasing FC could beattributed to the formation of smaller pores in the membranes. When theFC content was increased from 0.15 g to 0.25 g, the SEM pore diameterwas significantly reduced from 10-15 μm to 5-10 μm. FIG. 6 shows SEMmicrographs of top and cross-sectional views of the PS-FC membranes. Inparticular, FIGS. 6A (top) and 6B (cross-sectional) show images ofPS-FC-0.15; FIGS. 6C (top) and 6D (cross-sectional) show images ofPS-FC-0.20; and FIGS. 6E (top) and 6F (cross-sectional) show images ofPS-FC-0.25. The pore diameters as calculated based on Eq. 3 in the MWCOstudy were much smaller than those observed under SEM (see Table 1). Themeasurements from MWCO experiments most likely gave a more accuratedetermination of pore size than SEM measurements. For the commercial BTSmembranes, the SEM pore diameter was also found to be different than thepore diameter measured by MWCO.

In general, the PS-FC membranes showed a transition in permeationproperties from microfiltration to ultrafiltration range with increasingFC content. Their cut-off curves were sharper compared to that of thecommercial BTS-45 and BTS-55 PS membranes. These findings indicated thatthe PS-FC membranes are promising materials as porous separationmembranes.

The typical PEG rejection curves for the PS-FC membranes as a functionof PEG molecular weight are shown in FIG. 7. The cut-off level wasmeasured based on 90% rejection of PEG of a particular molecular weight.In general, the cut-off level of the membrane corresponded to its meanpore size.

The mean pore sizes were determined from the cut-off values as measuredat 90% rejection for PEG molecules. The increase in MWCO with decreasingFC content indicated an increase in pore size.

This example shows that membranes having uniform pore sizes can befabricated according to methods described herein.

Example 3 Growth of Cells on PS-FC Membranes

This example shows that the PS-FC membranes of Example 1 are compatiblewith living cells. For the biocompatibility study, MDCK (Madin-Darbykidney cells) were plated on the PS-FC membranes (without coating themembranes with cell adhesion proteins), and were cultured in ahumidified incubator with 5 vol % CO₂. The cell viability andproliferation were examined with optical fluorescent microscopy usingDAPI staining.

The morphology of the MDCK cells was studied after 4 days of culture ona PS-FC-0.25 membrane. FIG. 8 shows DAPI staining of the nuclei of theliving cells. FIGS. 8B and 8C shows that the MDCK cells adhered to themembrane, and covered the membrane surface homogeneously as a monolayer,as is characteristic of renal tubule cells, even without the use of celladhesion proteins. In contrast, FIG. 8A shows the adhesion of cells inthe form of clusters on a commercial polysulfone SUPOR_(—)1200 membrane(Pall Corporation, USA) when the membrane did not include a layer ofcell adhesion proteins. Thus, it was concluded that the PS-FC membraneprovided a non-toxic substrate for a better culture of a monolayer ofMDCK cells, compared to the SUPOR_(—)1200 membrane.

This example shows that PS-FC membranes can serve as bioactive membraneswithout further processing, such as coating the membranes with celladhesion proteins. This represents an improvement compared to existingpolymer surfaces used for bioartificial purposes, since manyconventional polymer membranes fail to show cell adherence without ECMsurface coatings. This example also suggests that PS-FC materials may besuitable for application in biohybrid artificial organ devices.

Example 4

Fabrication of 3D Porous Structures

In this prophetic example, a 3D porous structure suitable for use as atemplate for cell growth is fabricated. A CT scan of a tissue and/ororgan of a patient is converted into a CAD file and fed into athree-dimensional printer. A structure precursor material is prepared bymixing Fullcure™ 700 monomer, polysulfone, and N,N-dimethylacetamidesolvent, to form a homogeneous solution. The structure precursormaterial is introduced into the three-dimensional printer, as is asacrificial material (e.g., Fullcure™ 705 support) for forming openareas (e.g., cavities) in the structure. The structure precursormaterial and sacrificial material are dispensed droplet by droplet andlayer by layer onto a substrate. After several layers of the precursorstructure are deposited, the precursor structure is subjected to UVradiation for a sufficient period of time to cause polymerization(and/or crosslinking) of the Fullcure™ monomer. The resulting precursorstructure is immersed in a water bath, which causes precipitation of thepolysulfone and removal of the N,N-dimethylacetamide solvent from thestructure. As a result of this process, a uniform network of pores isformed in the structure. The structure is then immersed in 25% tetramethyl ammonium hydroxide (TMAH) solution until the sacrificial materialhas been removed from the structure. The structure is then used as atemplate for cell growth where living cells can be immobilized andperform their normal physiological functions.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A method of fabricating a structure for use as a template for cellgrowth, comprising: dissolving at least first and second polymercomponents in a precursor solvent to form a structure precursormaterial; shaping the structure precursor material into a structuresuitable for use as a template for cell growth; crosslinking the firstpolymer component; and removing at least a portion of the precursorsolvent from the structure, thereby forming a plurality of pores in thestructure.
 2. A method as in claim 1, further comprising contacting thestructure with a precipitation medium and removing the portion of theprecursor solvent in the precipitation medium.
 3. A method as in claim2, wherein the precipitation medium is a solvent.
 4. A method as inclaim 2, wherein the precipitation medium is water.
 5. A method as inclaim 2, wherein the precipitation medium is air.
 6. A method as inclaim 1, wherein the precursor solvent is non-reactive with the firstand second polymer components.
 7. A method as in claim 2, wherein thesecond polymer component is substantially non-crosslinked after thecrosslinking step.
 8. A method as in claim 1, wherein the plurality ofpores have an average pore size of less than or equal to 20 micronsformed in at least a portion of a wall of the structure.
 9. (canceled)10. A method as in claim 1, wherein no more than about 5% of all poresdeviate in size from the average pore size of the plurality of pores bymore than about 20%.
 11. (canceled)
 12. A method as in claim 1, whereingreater than 90% of the pores have a molecular weight cutoff of about 80kDa. 13-14. (canceled)
 15. A method as in claim 1, wherein shaping thestructure precursor material comprises three-dimensional printing.
 16. Amethod as in claim 1, further comprising exposing the structure to anenvironment facilitating cell growth onto the structure.
 17. A method asin claim 1, further comprising exposing the structure to an environmentfacilitating cell ingrowth into pores of the structure. 18-19.(canceled)
 20. A method as in claim 1, wherein the first polymercomponent is an acrylic-based monomer.
 21. A method as in claim 1,wherein the second polymer component is a sulfone-based monomer.
 22. Amethod of fabricating a structure for use as a template for cell growth,comprising: providing a structure precursor material comprising at leastfirst, second, and third components; shaping the structure precursormaterial into a structure suitable for use as a template for cellgrowth; crosslinking the first component; precipitating the secondcomponent in a precipitation medium; and removing the third componentfrom the structure in the precipitation medium, thereby forming aplurality of pores in the structure. 23-33. (canceled)
 34. A method offabricating a structure for use as a template for cell growth,comprising: mixing at least first and second polymer components in aprecursor solvent to form a homogeneous structure precursor material,wherein the first and second polymer components and the precursorsolvent are miscible at 25 degrees Celsius and 1 atm; printing thestructure precursor material to form a three-dimensional structuresuitable for use as a template for cell growth; and removing theprecursor solvent from the structure, thereby forming a plurality ofpores in the structure. 35-41. (canceled)
 42. A method of fabricating astructure for use as a template for cell growth, comprising: forming acell growth template precursor structure comprising at least first andsecond polymer components and a fluid carrier; crosslinking the firstpolymer component thereby forming a self-supporting structure; removingat least a portion of the fluid carrier from the self-supportingstructure, thereby forming a plurality of pores in the structuresuitable for templated cell growth, wherein the porous structure isformed in a shape suitable for templated cell growth. 43-46. (canceled)47. An article for use as a template for cell growth, comprising: astructure comprising at least one wall defining a cavity; and aplurality of pores having an average pore size of less than or equal to20 microns formed in at least a portion of the wall, wherein no morethan about 5% of all pores deviate in size from the average pore size ofthe plurality of pores by more than about 20%, wherein the structure isconstructed and arranged for use as a template for cell growth. 48-56.(canceled)
 57. A method of fabricating a structure for use as a templatefor cell growth, comprising: dissolving at least first and secondpolymer components in a precursor solvent to form a structure precursormaterial; shaping the structure precursor material into a structuresuitable for use as a template for cell growth; exposing the structureprecursor material to UV radiation; and removing at least a portion ofthe precursor solvent from the structure, thereby forming a plurality ofpores in the structure. 58-60. (canceled)